Nucleic acid having promoter activity and transformed plant containing the same

ABSTRACT

A novel nucleic acid which can exert promoter activity in phloem tissue as well as a transformed plant containing the nucleic acid and that can express a desired structural gene controlled by the nucleic acid in its phloem tissue is disclosed. The nucleic acid of the present invention has the nucleotide sequence shown in SEQ ID NO: 1 or 2 in SEQUENCE LISTING or a nucleic acid having a nucleotide sequence which is the same as the nucleotide sequence shown in SEQ ID NO: 1 or 2 in SEQUENCE LISTING except that one or a plurality of nucleotides are substituted, deleted, inserted or added, the latter nucleic acid having a promoter activity in phloem tissue of a plant, or a nucleic acid which is a part of anyone of the nucleic acids, that has a promoter activity in phloem tissue of a plant. The transformed plant of the present invention is one transformed with the nucleic acid of the present invention and a desired structural gene which is functionally ligated to a site downstream of the nucleic acid and which is controlled by the nucleic acid as a promoter, which transformed plant expresses the structural gene in its phloem tissue.

[0001] This application is the national phase under 35 U.S.C. § 371 of PCT International Application No. PCT/JP99/04519 which has an International filing date of Aug. 23, 1999, which designated the United States of America. This Application is a continuation of U.S. patent application Ser. No. 09/530,014, filed Apr. 24, 2000, the entire contents of which are hereby incorporated by reference.

TECHNICAL FIELD

[0002] The present invention relates to a nucleic acid which exerts promoter activity in the phloem tissue of a plant, and to a transformed plant that contains the nucleic acid and that can express a desired foreign gene in the phloem tissue thereof.

BACKGROUND ART

[0003] It is known that phosphoenolpyruvate carboxykinase (PCK; EC4.1.1.49) is localized in the cytoplasms of bundle sheath cells and functions as an enzyme indispensable to C4 photosynthesis in the C4 plants of the type which utilizes the enzyme as the main decarboxylase (PCK type C4 plants). It is also known that the enzyme functions as a decarboxylase for C4 organic acids in CAM plants of the type which utilizes the enzyme as a decarboxylase (PCK type CAM plants). In C3 plants, a PCK relating to gluconeogenesis in germination of seeds is known, and in an algae, a PCK relating to concentration of carbon dioxide is known.

[0004] As for plant PCK genes, although cDNAs have been isolated from cucumber (C3 plant) (Dae-jae Kim et al., Plant Molecular Biology 26: 423-434, 1994) and Urochloa panicoides (Patrick M. Finnegan et al., Plant Molecular Biology 27: 365-376, 1995), respectively, the promoter regions thereof have not been reported. Further, none of these genes have been reported for their promoter activities in plants.

[0005] On the other hand, some promoters controlling the phloem-specific expression in plants have been reported (U.S. Pat. No. 5,495,007, German Patent No. 4,306,832, International Publication Nos. WO 9304177, WO 9222582, and WO 9109050). However, none of these are the promoter of PCK gene.

[0006] As the transformation technology develops, it is becomingly possible to create transformed plants into which exogenous characters are introduced. In cases where it is necessary to express an introduced gene tissue (cell)-specifically, it is necessary to use a promoter controlling the tissue (cell)-specific expression. For example, since transportation and translocation of metabolites and the like are carried out through sieve tubes, it is desired to express a gene which is introduced for the control of the translocation specifically in the vicinity of the sieve tubes. It is thought that isolation of a promoter gene which is strongly expressed in the vicinity of sieve tubes would be an effective tool for construction of transformed plants.

DISCLOSURE OF THE INVENTION

[0007] Accordingly, an object of the present invention is to provide a novel nucleic acid which exerts promoter activity in the phloem tissue of a plant, and a transformed plant that contains the nucleic acid and that can express a desired foreign gene in the phloem tissue thereof.

[0008] As a result of intensive study, the present inventor has succeeded, for the first time, in identifying the promoter of the PCK gene of Urochloa panicoides which is a C4 plant, and in sequencing the promoter. Further, the present inventor succeeded in actually preparing plants transformed with the promoter and the desired structural genes ligated to a site downstream of the promoter, thereby completing the present invention.

[0009] That is, the present invention provides a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1 or 2 in SEQUENCE LISTING or a nucleic acid having a nucleotide sequence which is the same as the nucleotide sequence shown in SEQ ID NO: 1 or 2 in SEQUENCE LISTING except that one or a plurality of nucleotides are substituted, deleted, inserted or added, the latter nucleic acid having a promoter activity in phloem tissue of a plant, or a nucleic acid which is a part of anyone of the nucleic acids, that has a promoter activity in phloem tissue of a plant. Further, the present invention provides a transformed plant transformed with the nucleic acid according to the present invention and a desired structural gene which is functionally ligated to a site downstream of the nucleic acid and which is controlled by the nucleic acid as a promoter, which transformed plant expresses the structural gene in its phloem tissue.

[0010] By the present invention, a novel nucleic acid which exerts promoter activity in the phloem tissue of a plant was provided. By using the nucleic acid of the present invention as a promoter, it was made possible to strongly express a target gene specifically in the vicinity of sieve tubes of a plant such as rice. The nucleic acid is also effective when it is desired to express the target gene also in glume, pollen and anther. The promoter having such a property is useful for expressing an insecticidal protein, anti-viral protein, disease resistant protein or the like, thereby giving disease resistance or insect resistance to plants. The promoter is also useful for expressing a protein (e.g., proteins regarding carbon metabolism and sugar transporters) for supplying a nutrient such as carbon source (saccharide) or nitrogen source from the source to the sink, or for expressing a protein for supplying a signal transducer from the sink to the source. Besides, the promoter is especially useful in cases where it is necessary to express a protein for the purpose of transporting a substance through sieve tubes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows the structures of recombinant vectors pSB4 PCK1 and pSB4 PCK3 constructed in Examples, and shows the method for constructing these recombinant vectors.

[0012]FIG. 2 shows the structures of recombinant vectors pSB4 PCK1 bar and pSB4 PCK3 bar constructed in Examples, and shows the method for constructing these recombinant vectors.

BEST MODE FOR CARRYING OUT THE INVENTION

[0013] By the method detailed in the Examples described below, the present inventor cloned two types of DNAs containing the promoter sequences of PCK genes of Urochloa panicoides and determined the nucleotide sequences thereof. The determined sequences are shown in SEQ ID NOs: 1 and 2 in SEQUENCE LISTING. The nucleic acids having these nucleotide sequences exert promoter activities specifically in the phloem tissue of plants (as described in Examples below, the nucleic acids exert promoter activities also in glume, pollen and anther).

[0014] In general, it is well-known in the art that there are cases wherein the physiological activity of a physiologically active nucleic acid is retained even if the nucleotide sequence of the nucleic acid is modified such that one or more nucleotides are substituted, deleted, inserted or added. Therefore, nucleic acids having the same nucleotide sequence as the SEQ ID NO: 1 or 2 except that the nucleotide sequences of the nucleic acids are modified such that one or more nucleotides are substituted, deleted, inserted or added, which have the promoter activities exerted in the phloem tissues of plants, are included within the scope of the present invention. It is preferred that the nucleotide sequences of these nucleic acids have homologies of not less than 70%, more preferably not less than 90%, still more preferably not less than 95%, with the nucleotide sequence shown in SEQ ID NO: 1 or 2. Further, it is preferred that these nucleic acids hybridize with the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1 or 2 under stringent conditions (i.e., hybridization is performed at 60 to 65° C. using a common hybridization solution such as 5× Denhardt's reagent, 6×SSC, 0.5% SDS).

[0015] Further, since it is known that promoter activities are exerted by nucleic acids with sizes of about 80 bp, parts, especially those with sizes of not less than about 80 bp, of the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1 or 2 in SEQUENCE LISTING or of a nucleic acid having a nucleotide sequence which is the same as the nucleotide sequence shown in SEQ ID NO: 1 or 2 in SEQUENCE LISTING except that one or a plurality of nucleotides are substituted, deleted, inserted or added, the latter nucleic acid having a promoter activity in phloem tissue of a plant, are included in the scope of the present invention.

[0016] Since the origin and the nucleotide sequence of the nucleic acids according to the present invention have been clarified, the nucleic acids according to the present invention may easily be prepared or isolated by PCR using the genomic DNA of Urochloa panicoides as a template. In the present specification, the term “isolation” means cutting out the nucleic acid fragment from the genome or the like so as to enable the nucleic acid fragment to be cloned, or amplifying the nucleic acid fragment by a nucleic acid-amplification method such as PCR, or synthesizing the nucleic acid fragment.

[0017] By transforming a plant with the nucleic acid having the promoter activity according to the present invention, and with a desired structural gene which is functionally ligated to a site downstream of the nucleic acid of the present invention and which is controlled by the nucleic acid as a promoter, the structural gene may be expressed in the phloem tissue of the plant. Methods of transformation of plants per se are well-known in the art. Examples of the methods include electroporation (Toriyama K. et al., 1988; Bio/Technol. 6:1072-1074, Shimamoto K. et al., 1989; Nature 338:274-276, Rhodes C. A. et al., 1989; Science 240:204-207), PEG Method (Zhang W. et al., 1988; Theor. Appl. Genet. 5 76:835-840, Datta S.K. et al., 1990; Bio/Technol. 8:736-740), particle gun method (Gordon-Kamm W.

[0018] J. et al., 1990; Plant Cell 2:603-618, Fromm M.E. et al., 1990; Bio/Technol. 8:833-839, Christou P. et al., 1991; Bio/Technol. 9:957-962) and the like. Among the methods for transformation, the agroinfection method via Agrobacterium is preferred since the reproducibility and efficiency are high, and the method may be applied to plants for which a regeneration method from protoplasts have not been established. The agroinfection method per se is well-known in the art and described in, for example, Japanese Patent No. 2649287 and U.S. Pat. No. 5,591,616, and vectors for agroinfection are commercially available. By incorporating the nucleic acid having the promoter activity according to the present invention and the desired structural gene to be expressed into such a commercially available vector, a recombinant vector according to the present invention may easily be prepared. In cases where the desired structural gene is large so that it is difficult to directly incorporate the structural gene into the vector, the recombinant vector may be prepared by incorporating the structural gene into a vector for E. coli , which vector has a region homologous to the vector for Agrobacterium, and then allowing homologous recombination between the vectors in Agrobacterium(Komari, T. et al., Plant J. (1996) 10:165-174). Introduction of a plasmid into Agrobacterium may be carried out by, for example, the well-known triple cross method (Ditta G. et al., 1980; Proc. Natl. Acad. Sci. USA 77:7347-7351; Komari, T et al., supra). In cases where the nucleic acid of the present invention having the promoter activity and the structural gene are incorporated into a vector for agroinfection, the nucleic acid and the structural gene downstream thereof are inserted between the right and left border sequences of the T-DNA. Further, in cases where a commercially available vector for Agrobacterium is used, the promoter region of the commercially available vector may be cut out and the nucleic acid having the promoter activity may be inserted into the site at which the original promoter region was located, as described in the Examples below. As the vector for Agrobacterium, the super binary vector (i.e., the vector containing the virulence region of the Ti plasmid pTiBo542 contained in Agrobacterium tumefaciens A281 and the right and left border sequences of the T-DNA of the Ti plasmid or Ri plasmid of a bacterium belonging to the genus Agrobacterium) described in Japanese Patent No. 2649287 may preferably be used. Transformation of plants by Agrobacterium into which the recombinant vector of the present invention was introduced may be carried out by the well-known methods such as the method described in Japanese Patent No. 2649287.

EXAMPLES

[0019] The present invention will now be described in more concretely by way of examples thereof However, the present invention is not restricted to the examples below.

Example 1

[0020] (1) Isolation of PCK Genomic Clone

[0021] DNAs were isolated from green leaves of Urochloa panicoides using phenol (Komari, T. Plant Science 60:223-229 (1989)). One hundred micrograms of the DNAs were subjected to partial digestion with a restriction enzyme Sau3AI (produced by Pharmacia). The resultant was concentrated by isopropanol precipitation, and the obtained concentrate was subjected to continuous density gradient centrifugation of NaCl with a concentration of 20% to 5% (W/V) (maximum 240,000×g, 25° C., 4 hours and 30 minutes), followed by fractioning the resultant into small fractions. The size of the DNA in each fraction was determined by agarose gel electrophoresis and the DNAs from 23 to 15 kb was ligated to the BamHI site of lambda Dash II vector (trademark, produced by Stratagene) and a genomic library was prepared using Giga Pack Gold package mix (trademark, produced by Stratagene). Using the 880 bp region upstream of the Kpnl site of PCK cDNA of Urochloa panicoides (Patrick M. Finnegan et al., supra)as a probe, the genomic library was screened by the conventional method (Sambrook,J., Fritsch, E. F. and Maniatis,T.: Molecular cloning: A laboratory manual, 2nd ed., Cold spring harbor laboratory press, Cold Spring Harbor N.Y. (1989)) to obtain a plurality of positive clones. The clones were sequenced by T7 Sequencing TM Kit (trademark, Pharmacia) or by 373A DNA Sequencer (Applied Biosystems) to obtain PCK1 and PCK3 genes having very high homologies with the reported PCK cDNA. Since the homologies between the exon regions of the both genes and the cDNA were extremely high, both genes were thought to be PCK genes (Table 1). The 5′ upstream region which is upstream of the translation initiation site of each gene was referred to as the promoter region. The determined nucleotide sequences of PCK1 and PCK3 are shown in SEQ ID NOs: 1 and 2, respectively. TABLE 1 Homology with PCK1 cDNA PCK1 EXON 2 EXON 3 100% (169/169) 100% (179/179) PCK3 EXON 4 EXON 6 89.6% (138/154) 93.0% (280/299)

[0022] (2) Construction of Vector for Checking PCK Promoter Activity

[0023] Using synthetic primers which were designed to give restriction sites at both ends and Takara EX Taq (trademark, Takara Shuzo Co., Ltd.), the promoter regions of PCK1 and PCK3 were amplified by PCR method. The nucleotide sequences of the used synthetic primers were as follows: PCK1 Forward Primer: 5′CGCGTAAGGTTGATCCTGGAGCTTTATTATG 3′ (SEQ ID NO:4) Reverse Primer: 5′GATAGGATCCTCGTTCGAGCGTCGTGC 3′ (SEQ ID NO:7) PCK3 Forward Primer: 5′GACGTAAGCTTGAGGAAGAGGTGGCTCTG 3′ (SEQ ID NO:6) Reverse Primer: 5′GATAGGATCCTCGTTCGAGCGTGTCCTGCA 3′ (SEQ ID NO:5)

[0024] As for PCK3, since a fragment having a size shorter than the expected size was obtained, the entire nucleotide sequence was determined (SEQ ID NO: 3) using 373A DNA Sequencer (Applied Biosystems). As for PCK1, since a fragment having the expected size was obtained and since the sites cut with the restriction enzyme had the same nucleotide sequences as the both ends, respectively, it was judged that the desired fragment was obtained.

[0025] Each of the amplified fragment was replaced with the 35S promoter of an intermediate vector pSB21 for Agrobacterium at the BamHI and HindIII sites, which intermediate vector was prepared by removing the introns in the GUS gene of pSB24 (Komari T. et al., supra). The resulting vector was introduced into E. coli LE392 and then introduction into Agrobacterium and homologous recombination (Komari, T. et al., supra) were carried out by triple cross between Agrobacterium LBA4404/pSB4 (Komari T. et al., supra) and E. coli HB101/pRK2013 (Komari T. et al., supra). The resulting vectors were named pSB4PCK1 and pSB4PCK3, respectively. The structures of these recombinant vectors are shown in FIG. 1. In FIG.1 and in FIG. 2 described below, both the PCK1 gene and PCK3 gene are designated “PCKpro”. Further, in FIGS. 1 and 2, each symbol has the following meaning:

[0026] BL: left border sequence of T-DNA of Agrobacterium

[0027] BR: right border sequence of T-DNA of Agrobacterium

[0028] TC: tetracycline resistant gene

[0029] SP: spectinomycin resistant gene

[0030] HPT: hygromycin resistant gene

[0031] bar: bar gene

[0032] COS, cos: COS site of λ phage

[0033] ORI, ori: replication origin

[0034] 35Spro: 35S promoter of cauliflower mosaic virus (CaMV)

[0035] NOSter: terminator of nopaline synthetase

[0036] virB: virB gene of Ti plasmid pTiBo542 contained in Agrobacterium tumefaciens A281

[0037] virC: virc gene of Ti plasmid pTiBo542 contained in Agrobacterium tumefaciens A281

[0038] virG: virG gene of Ti plasmid pTiBo542 contained in Agrobacterium tumefaciens A281

[0039] Vir: entire vir regions of Ti plasmid of Agrobacterium

[0040] GUS: β-glucuronidase gene

[0041] (3) Preparation of Transformed Rice by Agrobacterium method

[0042] Using the Agrobacterium into which the recombinant vector was introduced as mentioned above, and using Japonica rice variety “Tsukinohikari” as the plant to be transformed, transformation was performed by the Agrobacterium method according to the reported method (Hiei, Y. et al., (1994) Plant J. 6: 271-282). The obtained transformants were grown in an air-conditioned green house (16 hours daylight, day: 28° C., night: 23° C.).

[0043] (4) Identification of Expressing Tissue by GUS Staining and Measurement of GUS Activity of Green Leaves

[0044] Small sections of the leaves and roots of regenerated young plants were immersed in 50 mM phosphate buffer (pH 7) containing 20% methanol and 1 mM 4-methylumbelliferyl-β-D-glucuronide and incubated therein overnight at 37° C., thereby carrying out GUS staining. The plants which showed GUS expression were transplanted to pots. (As a negative control, one plant which did not show expression was also transplanted to a pot). The β-glucuronidase activity of the green leaves of the transformed plants grown in an air-conditioned green house (day: 28° C./night: 23° C., 16 hours daylight) was measured by the method of Matsuoka et al. (Makoto Matsuoka and Yasuharu Sanada (1991) Mol Gen Genet 225: 411-419) with the fluorescent light of 4-methylumbelliferyl-β-D-glucuronide (4 MU). The amount of the protein was measured by using BioRad Protein Assay (trademark, Japan BioRad Laboratories). Tissue-specific expression was checked for sections of green leaves, stems and roots, and for glumes by the method of Matsuoka et al. (Makoto Matsuoka and Yasuharu Sanada (1991) Mol Gen Genet 225: 411-419). The sections were prepared using a microslicer DTK-2000(produced by Dohan E M) under the conditions called speed 2, frequency 7, and the GUS staining reaction was carried out overnight at 37° C.

[0045] (5) Results

[0046] Green leaves of 6 GUS staining-positive plants among the transformed plants into which pSB4PCK1 was introduced, 5 GUS staining-positive plants among the transformed plants into which pSB4PCK3 was introduced, and of one plant which was GUS staining-negative were checked for the GUS activities. As a result, plants showing high GUS activities were obtained for both transformants (Table 2). Sections of green leaves, stems and of roots, as well as glumes were checked for the expression of GUS, and strong expression was observed in the vicinity of sieve tubes from the leaves to the roots, but expression was not observed in other tissues containing photosynthesis cells. Expression was also observed in the hairs on the surfaces of glumes, pollen and anthers (Table 3). Clear difference was not observed in the tissue specificity of expression pattern between the transformed rice plants into which pSB4PCK1 and pSB4PCK3 were introduced, respectively, although the intensities of staining were different.

[0047] Based on the above-described results, it was thought that the gene fragments of PCK1 and PCK3 of Urochloa panicoides have promoter activities which express genes specifically in the vicinity of sieve tubes (from roots to leaves through stems) of C3 plants (at least rice). Further, it was confirmed that the gene fragments have promoter activities which express genes in glumes, pollen and anthers. TABLE 2 GUS Activity in Green Leaves (pmol/min/mg protein) PCK1 PCK3 Control 1 1013 39750 12.4 2 934 6476 3 2053 261 4 8849 597 5 380 5152 6 2952

[0048] TABLE 3 Tissues Which Showed GUS Expression Leaf Blade Cells in large and small vascular bundles (especially the region sandwiched between sieve tubes and vessels) Leaf Sheath Cells in large and small vascular bundles (especially the region sandwiched between sieve tubes and vessels) Stem Nodal net vascular bundles, circumferencial vascular bundle ring, root rudiment Root Cells in large and small vascular bundles Ear Surfaces and hairs of lemma and palet, rachis-branch Anther Filament vascular bundles, pollen

Example 2

[0049] (1) Construction of Vector for Maize

[0050] The 35S-bar-nos fragment with a size of 2.2 kb which was cut out from pSB25 (described in WO 95/06722) by digestion with HindIII and EcoRI was inserted into the HindIII site of pSB21 PCK which was constructed for rice. The obtained vector was introduced into E. coli LE392, and introduction to Agrobacterium and the homologous recombination (Komari, T. et al., Plant J. (1996) 10: 165-174) were carried out by triple cross between Agrobacterium LBA4404/pSB1 and E. coli HB101/pRK2013 (FIG. 2). The resulting vectors were named pSB4PCK1 bar and pSB4PCK3bar.

[0051] (2) Transformation of Maize

[0052] Transformation of maize was carried out in accordance with the reported method (Y. Ishida, S. Saito, S. Ohta, Y. Hiei, T. Komari and T. Kumashiro (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotechnology 14:745-750) using Agrobacterium.

[0053] (3) Identification of Expressing tissue by GUS Staining and Measurement of GUS Activity

[0054] GUS staining of green leaves and roots of transformed maize were carried out by immersing them in 50 mM phosphate buffer (pH 7) containing 20% methanol and I mM 4-methylumbelliferyl-β-D-glucuronide overnight at 37° C. To check the cell-specific expression, sections were prepared using a microslicer DTK-2000(produced by Dohan E M) under the conditions called speed 2, frequency 7. The β-glucuronidase activity was measured in accordance with the method by Matsuoka et al. (Makoto Matsuoka and Yasuharu Sanada (1991) Mol Gen Genet 225: 411-419) with the fluorescence of 4-methyumbelliferyl-β-D-glucuronide (4 MU). The amount of the protein was measured by using BioRad Protein Assay (trademark, Japan BioRad Laboratories).

[0055] (5) Results

[0056] Plants showing GUS activities in green leaves and roots were obtained from both transformants to which pSB1PCK1bar and pSB1PCK3bar were introduced, respectively. Tissues were subjected to GUS staining, and specific expression in green leaves and in the vascular bundles in roots was observed. Clear difference was not observed in the expression sites between the PCK1 and PCK3 promoters. These results were similar to those obtained for rice.

[0057] These results prove that PCK1 and PCK3 show promoter activities specifically in the vascular bundles of not only C3 plants, but also of C4 plants. TABLE 4 GUS Activity (pmol/min/mg protein) of Present Generation (R0) of Transformants PCK1 PCK3 No. Green Leaf Root Green Leaf Root 1 132 60 958 121 2 2220 206 1170 122 3 699 41 173 97 4 530 119 376 74 5 2010 170 173 140 6 1270 85 458 107 7 10201 1225 386 84 8 4004 1021 3471 227 9 669 ND 1602 ND 10 5605 ND 11 1933 ND 12 1290 ND 13 4814 ND 14 994 ND 15 3040 ND 16 10859 ND 17 360 ND 18 167 ND 19 2834 ND 20 5111 ND 21 9848 ND

[0058]

1 7 1 1327 DNA Urochloa panicoides 1 gatcctggag ctttattatg tttatttgga aacttgcatc tctaatttct ttttacatga 60 agcattctat gcatctctaa tttttgcccc ggaccaaaca accctttata atacttggtg 120 atgtattctt aaggttgtct tcgcctacta tgactccctc cttccggtca agcctaaaaa 180 ttctagtttt ctttctttta ccatttgcta ccagctgaaa gtatttggta ttgttgtctc 240 cttgtaagag ctctatagtt ttaaatcttt gaaaccactt tatttcttcc tctttaagga 300 gttgtaccgg tctatctttt atacattgtt ttaaatcaat ttactgctgc gttagaagct 360 gagattcatc tttgtttatc taactcattc gccttcctaa gcagctcttg cttttccttc 420 ttgtatgccc cgttgatatt cttagcccaa cctccccgaa actgtctcag tctcctaatt 480 ttgttttgcc atttttgctt ggctgtggaa ccttttattt tctctgttcc aaatttctag 540 ttttcttaac atcttcctcc ctccgcaaaa acttggctag ccttttggta gcatgcaaac 600 aagtgtacct tgccccccac aaagtaagat gacaagttgg tctattgtta ttgtccgttc 660 tattatttat ttgaaagaat taatttcttt accttttttt tcaatcatta tctatgtgtg 720 tggaggaagt gctgaagaat cgaccaattt gatgcttcaa acaagtatgc aaagccagaa 780 ttagatggtt gcggccgtga tgcacaattg taatgcaaca cttgaaaatg aactcgatct 840 ggtgtctagt taccaacgcc gacagctgaa aatattcttg cagtacgtcc tgcatagagg 900 cagggaaaca gaggtgagaa aaaaaaaagc tccagatttc caattccaaa cccaacccgg 960 catcgcagtg tggtcaggta gagagcgtaa ccacggttaa ctcaactctg gggcgtcctt 1020 taaaagccca gccgggctcg tcccatccgc cccacacagc ttcactggtc tcctttcagt 1080 tccaactcca caccgcttcc tcggcggccg gcgcacgtcg ctgctcccgt acgtgcccgc 1140 ctctctcctc ctcctcgtgt tcaatccaat cccggcccgc tgcaaaatcc tcgcagcgac 1200 gccctgctga tgctctcgaa gctagcgtca atggcggccg tgatcgtggc tctactagtc 1260 tccctaggct gctatataat gcgcgcgctc tcgctgtgtt ttccctcttg caggacgctc 1320 gaacgag 1327 2 1973 DNA Urochloa panicoides 2 ggaagaggtg gctctgttac catgtagaat tctatggaac ggcttacccc aatgagggcg 60 ccggtgtttc attatatagg ccgggatatg accgacagag tcctagtagg attagaagta 120 ttacatgagt aggactcaat aagtccatat cctatacaat gagaactata tctcttacag 180 acagctcaat ggaaccatat tttttgttaa aaagaaaaag aaaatgcaat gattttccgt 240 gatcacaatt cacagcagct tttttattta gaatacaaac tggaatcatg tatatgatga 300 caagctggtc cattattact gtccattcca ttatttgaag gaattacttt cttcaccttc 360 tctcaataca ttttctagat ctagagttaa atacactaaa ggtatataaa cttgtcctga 420 ggtgccactt aggtgcatac ccttcgatat atgcaaaaga cacctcaagt tttaatcttc 480 tacatctagg tccttctctt ctctctcttg tctttctttc tcaaccgagt accacgacgc 540 cagccatacc ctgatctctg ctccaccgcc agcgcggacc ggctccccct ccctccgcca 600 gctcgcggga ccggcagccg gagcacccgg gctcgggacc tcgcagaggt gcgcggtgcc 660 gcatcggcga gcgggctgcc tcggggagtg gcgcggccgg cgtagctccc acgcagcatg 720 gccggcgagc tcaacgcaga ggagacttcc tccagtgccg gacatggtgt gatgaagcct 780 cgaattgact cgcacgggcc tcgaatcgac gtcgcgaagc ttgaccccga ccttctcctt 840 tcccagcctc ctctccccac ctccgccggg ggaccagccg cctgcacacc ggagcggaaa 900 cgacggcggg ccacacacgt ccctgaccga ggcgcgggag gtggagctca agcgcttgcg 960 ggtcacgcgt gtcccttgcc gaggctcggg aggtggagct ctgacgcacc ggcccccgta 1020 gagaagagtg tcgccgcggg gatggactcc ttctccctgc cctcgatttg tccgtcacgg 1080 tagagttcct tcaggcgagg gaggaggagg catggtggtg gagctcatgc tgctctgcgc 1140 gacggcgaca aggagcagcg accgatcttg gccctcatcc ctccccacgt agacaagaag 1200 gacttagaca tataaaaaga ttaaaactcg aggtgtcttt tacatatatc gaaggggtgt 1260 ggacctaagt ggcacaactt tacaagtttt ggaagttgga tgtgcatttt tcaagttcgt 1320 ggacctaagt aacaccttag gacaagttta tgtaccctga tgtatttaac tctattttct 1380 atgtgcgggg aggaagtggt gaagaatcga ccaatttgat ttgatgcttc agacaagtac 1440 tgaaagcagg aatcatatgg cgggccgtgc tgtagaattg tgctgccaca ctgaaaatca 1500 acctgaaaac atcctgcagt acgtcctgcc tagagagtta gagacagaga aacagagtgg 1560 agaaaaaaaa atcaaagaaa caagctccag agttccaact ccaaacccaa cccggcacca 1620 cagtagtcag gtagcgaacg taaccacggt taacttaact cccgggcagc ctttaaaagc 1680 ccagcccggc tcgtcccatc cgccccacac agtttcactg gtttcctttc gattccaaca 1740 ccacaccgct tcctcggcgc acgtcgttgc tcccgtacgt ccccgtctct cctcctcctc 1800 gtgtccagtt caatcgatct cggcccgttg caatcctcgc agctacgccc tgatgctctc 1860 caagctagcg tcaatggcgg ccgtgatcgt ggtgctcttc ctagctaggc tgctagctaa 1920 tgcgcggcgc tcgctgtgtt tttccccccc tttgcaggac acgctcgaac gag 1973 3 1710 DNA Urochloa panicoides 3 gcttgaggaa gaggtggctc tgttaccatg tagaattcta tggaacggct taccccaatg 60 agggcgccgg tgtttcattg tataggccgg gatatgaccg acagagtcct agtaggatta 120 gaagtattac atgagtagga ctcaataagt ccatatccta tacaatgaga actatatctc 180 ttacagacag ctcaatggaa ccatattttt tgttaaaaag aaaagaaaaa tgcaatgatt 240 ttccgtgatc acaattcaca gcagcttttt tatttagaat acaaactgga atcatgtata 300 tgatgacaag ctggtccatt attactgtcc attccattat ttgaaggaat tactttcttc 360 accttctctc aatacatttt ctagagttaa atacactaaa ggtatataaa cttgtcctga 420 ggtgccactt aggtgcatac ccttcgatat atgcaaaaga cacctcaagt tttaatcttc 480 tacatctagg tccttctctt ctctctcttg tctttctttc tcaaccgagt accacgacgc 540 cagccatacc ctgatctctg ctccaccgcc agcgcggacc ggctccccct ccctccgcca 600 gctcgcggga ccagccgcct gcacaccgga gcggaaacga cggcgggcca cacacgtccc 660 tgaccgaggc gcgggaggtg gagctcaagc gcttgcgggc cacgcgtgtc ccttgccgag 720 gctcgggagg cggagctctg acgcaccggc ccccgtagag aggagtgccg ccgcggggat 780 ggactccttc tccctgccct cgatttgtcc gtcacggtag agttccttca ggcgagggag 840 gaggaggcat ggtggtggag ctcatgctgc tctgcgcgac ggcgacaagg agcagcgacc 900 gatcttggcc ctcatccctc cccacgtaga caagaaggac ttagacatat aaaaagatta 960 aaactcgagg tgtcttttac atatatcgaa ggggtgtgga cctaagtggc acaactttac 1020 aagttttgga agttggatgt gcatttttca agttcgtgga cctaagtaac accttaggac 1080 aagtttatgt accctgatgt atttaactct attttctatg tgcggggagg aagtggtgaa 1140 gaatcgacca atttgatttg atgcttcaga caagtactga aagcaggaat catatggcgg 1200 gccgtgctgt agaattgtgc tgccacactg aaaatcaacc tgaaaacatc ctgcagtacg 1260 tcctgcctag agagttagag acagagaaac agagtggaga aaaaaaaatc aaagaaacaa 1320 gctccagagt tccaactcca aacccaaccc ggcaccacag tagtcaggta gcgaacgtaa 1380 ccacggttaa cttaactccc gggcagcctt taaaagccca gcccggctcg tcccatccgc 1440 cccacacagc ttcactggtt tcctttcgat tccaacacca caccgcttcc tcggcgcacg 1500 tcgttgctcc cgtacgtccc cgtctctcct cctcctcgtg tccagttcaa tcgatctcgg 1560 cccgttgcaa tcctcgcagc tacgccctga tgctctccaa gctagcgtca atggcggccg 1620 tgatcgtggt gctcttccta gctaggctgc tagctaatgc gcggcgctcg ctgtgttttt 1680 cccccccttt gcaggacacg ctcgaacgag 1710 4 31 DNA Artificial Synthetic primer derived from Urochloa panicoides 4 cgcgtaagct tgatcctgga gctttattat g 31 5 30 DNA Artificial Synthetic primer derived from Urochloa panicoides 5 gataggatcc tcgttcgagc gtgtcctgca 30 6 29 DNA Artificial Synthetic primer derived from Urochloa panicoides 6 gacgtaagct tgaggaagag gtggctctg 29 7 27 DNA Artificial Synthetic primer derived from Urochloa panicoides 7 gataggatcc tcgttcgagc gtcctgc 27 

1. (cancelled)
 2. A nucleic acid having the nucleic acid sequence shown in SEQ ID NO:1 or a nucleic acid which hybridizes with the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions and which has a promoter activity in phloem tissue of a plant.
 3. A nucleic acid having the nucleic acid sequence shown in SEQ ID NO:1 or a nucleic acid having a homology of not less than 70% with the nucleotide sequence shown in SEQ ID NO: 1 which has a promoter activity in phloem tissue of a plant.
 4. (cancelled)
 5. (cancelled)
 6. A nucleic acid having the nucleic acid sequence shown in SEQ ID NO:2 or a nucleic acid which hybridizes with the nucleic acid having the nucleotide sequence shown in SEQ ID NO: 2 under stringent conditions and which has a promoter activity in phloem tissue of a plant.
 7. A nucleic acid having the nucleic acid sequence shown in SEQ ID NO:2 or a nucleic acid having a homology of not less than 70% with the nucleotide sequence shown in SEQ ID NO: 2 which has a promoter activity in phloem tissue of a plant.
 8. (cancelled)
 9. A recombinant vector comprising the nucleic acid according to any one of claims 2, 3, 6, and
 7. 10. The recombinant vector according to claim 9, further comprising a desired structural gene which is functionally ligated to a site downstream of said nucleic acid and which is controlled by said nucleic acid as a promoter.
 11. A transformed plant transformed with said nucleic acid according to any one of claims 2, 3, 6, and 7 and a desired structural gene which is functionally ligated to a site downstream of said nucleic acid and which is controlled by said nucleic acid as a promoter, which transformed plant expresses said structural gene in its phloem tissue.
 12. The transformed plant according to claim 11, which belongs to the family Gramineae.
 13. The transformed plant according to claim 12, which is rice or maize.
 14. A nucleic acid having the nucleic acid sequence shown in SEQ ID NO:
 1. 15. A nucleic acid having the nucleic acid sequence shown in SEQ ID NO:2. 